Acetazolamide-based fungal chitinase inhibitors

Bioorganic & Medicinal Chemistry 18 (2010) 8334–8340
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acetazolamide-based fungal chitinase inhibitors
Alexander W. Schüttelkopf , Ludovic Gros , David E. Blair , Julie A. Frearson, Daan M. F. van Aalten ⇑,
Ian H. Gilbert ⇑
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee DD1 5EH, UK
a r t i c l e
i n f o
Article history:
Received 20 May 2010
Revised 21 September 2010
Accepted 24 September 2010
Available online 8 October 2010
Keywords:
Chitinase
Aspergillus fumigatus
a b s t r a c t
Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential
strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two
distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the
inhibition of an A. fumigatus ‘plant-type’ chitinase. A co-crystal structure of AfChiA1 with acetazolamide
was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement
with these structural data. Although acetazolamide and its analogues are weak inhibitors of the
enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor
development.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Aspergillus fumigatus is the causative agent of aspergillosis, a
life-threatening fungal infection that targets a rising population
of immunocompromised patients.1 Currently available anti-fungal
drugs, such as the azoles, amphotericin B and the candins are only
partially effective2,3 and resistant Aspergillus strains have started to
appear in hospital settings.4,5 Thus there is a need for the identification of novel targets and the development of new anti-fungal
agents. Enzymes involved in the biogenesis/turnover of the fungal
cell wall are thought to represent possible targets.
Chitin, a polymer of b(1,4)-linked N-acetylglucosamine
(GlcNAc), is an essential structural component of the fungal cell
wall, giving it structural rigidity and chemical/biological stability.
Because of the inherent rigidity of chitin, fungi need to partially
hydrolyse the chitin layer for cell division and morphogenesis,
which is carried out by family 18 chitinases.6 Two subclasses of
family 18 chitinases exist: the ‘bacterial-type’ chitinases are found
in bacteria, fungi and mammals; the ‘plant-type’ chitinases are
found exclusively in plants and fungi. Whereas the ‘bacterial-type’
enzymes are invariably secreted and mostly possess exochitinase
activity,7–10 the ‘plant-type’ chitinases are frequently cell wall
associated and possess endochitinase activity. Several studies have
shown that these enzymes are involved in yeast mother–daughter
⇑ Corresponding authors. Tel.: +44 1382 386 240; fax: +44 1382 386 373 (I.H.G.).
E-mail addresses: d.m.f.vanaalten@dundee.ac.uk (D.M.F. van Aalten), i.h.gilbert@dundee.ac.uk (I.H. Gilbert).
These authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.062
cell separation.11,12 Because these enzymes are not intracellular, it
is possible to explore a wider area of chemical space for inhibitors,
as these would not be required to cross membranes. Whilst
humans possess two active chitinases,13,14 they are of the ‘bacterial-type’, and to date the only inhibitors reported are the large,
hydrophilic natural products, allosamidin,15 argifin,16 argadin17
and the rationally designed drug-like inhibitor C2-dicaffeine.18
There are five ‘plant-type’ chitinases genes in the A. fumigatus genome (AfChiA1–5), with a currently unknown transcription profile.
Sequence alignments show that they have a high degree of structural similarity in the active site, suggesting that it should be possible to design compounds that inhibit all five enzymes.
Recently we have cloned and over-expressed the ‘plant-type’
family 18 chitinase Cts1p from Saccharomyces cerevisiae (ScCTS1).19
This enzyme was then screened against the Prestwick chemical
library of 880 drug-like molecules. From this, three significant hits
were identified, 8-chlorotheophylline, acetazolamide and kinetin
(Table 1), all of which were competitive inhibitors of the enzyme
and were shown to bind in the active site groove, interacting with
the catalytic machinery.19
Here we describe a study towards the identification of small
inhibitor scaffolds (‘fragments’) against the ‘plant-type’ A. fumigatus enzyme chitinase A1 (AfChiA1). Two novel ScCTS1 inhibitors,
acetazolamide and 8-chlorotheophylline, showed weak inhibition
of AfChiA1. We were able to obtain a crystal structure of AfChiA1
in complex with acetazolamide. A number of derivatives of acetazolamide were prepared or purchased and screened against the
enzyme AfChiA1; a number were identified with similar activity
to acetazolamide.
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Table 1
Inhibitors of ScCTS1 and their activity against AfChiA1
Name
Allosamidin
8-Chloro-theophylline
OH
HO
Structure
HO
O
O
OH
O
NHAc
HO
O
ScCST1 Ki
AfChiA1 IC50
hCHT IC50
N
O
OH
O
NHAc
HO
N
N
N
H
Cl
Acetazolamide
O
S
H2N S
O N N
Kinetin
H
N
H
N
O
N
N
N
O
HN
O
N
NMe2
0.61
127
0.04
340
410
>2500
21
164
>1000
3.2
>1000
>1000
All data given in lM. hCHT is human chitinase. Data for ScCST1 have been reported previously.19 The IC50 of allosamidin against hCHT has been reported previously.22
2. Results and discussion
2.1. Acetazolamide is an efficient inhibitor of A. fumigatus
chitinase
Previous work has suggested that the plant-type fungal chitinases may be targets for novel anti-fungal strategies.11,12,20 So far the
only enzyme from this class characterised in some detail is CST1
from S. cerevisiae and the plant enzyme hevamine.6 A. fumigatus chitinase A1 (AfChiA1) also belongs to the class of plant-type chitinases
family, and has been cloned and characterised recently.21
To identify possible inhibitors of AfChiA1, a number of planttype chitinase inhibitors previously characterised against ScCTS1
were explored as potential scaffolds (Table 1).19 Allosamidin is an
extensively characterised natural product inhibitor of both planttype and bacterial-type family 18 chitinases,15 and has recently
been reported to competitively inhibit ScCTS1 with a Ki of
0.61 lM19 and hevamine with a Ki of 3.1 lM.9 Unfortunately, allosamidin is a substrate analogue with poor drug-like properties (high
molecular weight, containing glycosidic bonds and an undesirably
low C log P of 5.2) and the total synthesis is costly and complicated.23 Remarkably, allosamidin only weakly inhibits AfChiA1
(IC50 = 127 lM, Fig. 1),21 which is 30- and 200-fold less potent than
values previously reported against hevamine and ScCTS1, respectively. Examination of the purine derivative 8-chlorotheophylline,
which had previously been demonstrated to inhibit ScCTS1 with
a Ki of 600 lM,19 revealed a similar level of inhibition (IC50 =
410 lM, Fig. 1). Two further compounds, kinetin and acetazolamide, have been identified as ScCTS1 inhibitors by screening the
Prestwick Chemical Library, with Ki values of 3.2 lM and 21 lM,
respectively.19 Kinetin failed to show any discernable effect against
AfChiA1 even at concentrations in excess of 1 mM; acetazolamide
on the other hand inhibited AfChiA1 with an IC50 of 164 lM
(Fig. 1), which is an order of magnitude less potent than previously
demonstrated against ScCTS1 but not dissimilar to the level of inhibition observed for allosamidin (Fig. 1). It is instructive to compare
the ligand efficiencies of the compounds at this stage—that is, the
binding energy per non-hydrogen atom.24 Due to their small size
acetazolamide and 8-chlorotheophylline are the most efficient of
these inhibitors (0.61 and 0.57 kcal mol1 atom1, respectively), compared to allosamidin (0.19 kcal mol1 atom1). Thus,
acetazolamide is a small drug-like molecule that is amenable to
preparation of analogues and represents an attractive starting
point for further elaboration.
2.2. Crystal structure of the AfChiA1–acetazolamide complex
suggests possible derivatives
Of the initial leads investigated, acetazolamide was selected
as the most promising starting point for the development of A.
fumigatus plant-type chitinase inhibitors given its high ligand
efficiency. AfChiA1 crystals, reported previously21, were soaked
with acetazolamide, diffraction data were collected to 2.0 Å resolution, and the structure of the AfChiA1–acetazolamide complex
was solved by molecular replacement and refined to an Rfree of
0.249 (Table 2) with good stereochemistry. Electron density for
the ligand acetazolamide can be seen in both molecules in the
asymmetric unit, but it is less clear in chain A, where the active
site is partially occluded by a symmetry-related protein molecule. Thus the further discussion of the structure will focus on
Figure 1. IC50 curves determined in triplicate, fitted to a four-parameter logistic dose–response curve (minimum, Hill slope, inflection point and maximum) against AfChiA1
for allosamidin (IC50 = 128 lM, Hill slope 0.9), acetazolamide (IC50 = 164 lM, Hill slope 1.1) and 8-chlorotheophylline (IC50 = 410 lM, Hill slope 1.0) using 4methylumbelliferyl b-D-N,N0 ,N00 -triacetylchitotrioside (4MU-NAG3) as a substrate.
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Table 2
X-ray diffraction/refinement statistics for the AfChiA1–acetazolamide complex
Resolution range (Å)
Number of observed reflections
Number of unique reflections
Completeness (%)
Redundancy
I/r(I)
Rmerge
Wilson B (Å2)
Rwork, Rfree
Bond length rmsd from ideality (Å)
Bond angle rmsd from ideality (°)
<B>, overall (Å2)
<B>, protein (Å2)
<B>, solvent (Å2)
<B>, ligand (Å2)
Ramachandran plot
Most favoured (%)
Additionally allowed (%)
Generously allowed (%)
20.00–2.00 (2.05–2.00)
27,0663
67,879 (4270)
98.1 (92.9)
4.0 (3.5)
13.5 (2.5)
0.085 (0.522)
22.5
0.216, 0.249
0.017
1.5
27.2
26.5
34.1
32.1
88.6
10.8
00.2
Values in parentheses pertain to the highest resolution shell. Ramachandran plot
statistics were calculated with PROCHECK.25
chain B only, which is less impacted and has a more accessible
active site.
The overall binding mode of the ligand to AfChiA1 is essentially
identical to that observed for ScCTS1 (Fig. 2B).19 The thiadiazole ring
stacks with the conserved Trp312, while its ring nitrogens accept
hydrogen bonds from the backbone amides of Ala124 and Tyr125,
in the latter case indirectly via an active site-bound water molecule.
The acetamido group enters, and essentially fills, the small AfChiA1
active site pocket formed by Tyr238, Gln230, Met310, Ala205, Tyr34
and Asp172. It is oriented by two hydrogen bonds, one from its
amide to the side chain of Asp172 and one from the Tyr232 side
chain hydroxyl to its carbonyl oxygen. The sulfonamide group on
the other hand forms few direct interactions with the protein: it accepts a poor hydrogen bond from the Trp312 side chain and otherwise points away from the protein and into the bulk solvent.
The unexpectedly poor inhibition of AfChiA1 by kinetin can be
explained by the presence of methionine 310 in the AfChiA1 active
site, which replaces an alanine in the corresponding position in
ScCTS1 (Fig. 2A), a substitution found in all A. fumigatus plant-type
chitinases. These residues define the bottom of the active site
pocket that accepts the furanyl group of kinetin.19 While the pocket is still present in AfChiA1, it is shallower due to the larger
Met310 side chain (Fig. 2), rendering it unable to accommodate
bulky ligands like kinetin.
A. fumigatus is predicted to possess five plant-type GH18 chitinases (AfChiA1–5) that may have overlapping, if not interchangeable,
functions. Thus, for an inhibitor to be useful in vivo, it would have to
bind effectively to all five AfChiA active sites. The AfChiA1 sequence
in Fig. 2A is shaded based on a sequence alignment of AfChiA1–5,
indicating residues identical among all five proteins in purple, residues conserved among four or fewer proteins in shades of blue and
completely non-conserved residues in white. The same colouring
has also been applied to the AfChiA1 active site surface shown in
Fig. 2C, demonstrating that, with the exception of the non-conserved but flexible Tyr125,21 the part of the active site cleft interacting with acetazolamide is completely conserved among all five A.
fumigatus plant-type chitinases. This suggests that acetazolamide
could bind similarly, both in orientation and in affinity, to these five
enzymes. Fig. 2C also highlights additional conserved active site
areas that could be used for the further elaboration of the ligand.
To investigate in silico the potential for such elaboration, we
used the docking program LIGTOR18 to screen for beneficial substitutions/modifications of either the acetamido or the sulfonamide
group, while keeping the rest of the molecule constant. Not surprisingly, the scope for modification at the acetamido group is limited.
Docking runs predict that a slight increase in size of this group, for
example, by substituting a trifluoroacetamido moiety, could improve overall binding affinity, and even an additional methyl group,
yielding a propionamido group, may be tolerated with slight
changes to the overall binding mode, but anything larger (including,
e.g., isobutyramido groups) cannot be accommodated in the active
site pocket and would most likely abolish binding. Modifications/
substitutions of the sulphonamide group on the other hand face
the opposite problem: as the ligand is essentially pointing away
from the active site, most small modifications are tolerated but do
not yield additional interactions between ligand and protein. Larger
additions to the existing scaffold may be able to interact with additional parts of the AfChiA1 active site, but the required flexibility of
such ligands and the corresponding entropy cost associated with
orienting the flexible parts on binding to the protein could negate
any positive effects on the predicted ligand affinity. To test these
computational predictions, a number of acetazolamide derivatives
were either synthesised or obtained from commercial suppliers
and their binding to AfChiA1 was investigated.
2.3. Synthesis and screening of acetazolamide derivatives
As acetozolamide provided an attractive small molecule starting
point for a rational focused inhibitor screen, including a structurally
defined binding mode, a number of analogues were screened
against AfChiA1 (Table 3). The acetazolamide analogues were either
synthesised (Scheme 1) or acquired from commercial sources. The
synthesis of compounds carried out to is shown in the scheme.
Compounds were screened against AfChiA1 in duplicate. The assay performance statistics generated from screening plates were
well within acceptable screening parameters (Z0 0.72 ± 0.04) and
the replicate potency determinations correlated well, yielding errors below 45% for all bar one compound (Table 3).
The structures of the compounds allowed determination of the
effects of changing the both the sulphonamide (R1 in Table 3) and
acetamide (R2) portions of the molecule. The screen gave a number
of compounds with potencies in the 100–500 lM range, that is,
similar to the parent compound. A few trends in the SAR can be deduced. Increasing the size of the acetamide moiety by adding an
extra methyl (2) or a chloro (20 and 21) substituent leads to a
reduction in activity, while substitution with a trifluoroacetamide
group (compare 11 and 15) is energetically neutral or slightly
favourable; this is in accordance with the structural and docking
data, as the methyl of the acetamide group essentially fills the active site pocket as described above. At the same time ‘deacetylating’ R2 to a free amine also abolishes inhibitory activity (cf. 6).
Replacement of the sulphonamide is generally tolerated: –SH, –
Ph, –CF3 and –Br substituents as R1 (9–12) produce compounds
with similar activity to acetazolamide (1). This is perhaps not surprising as the sulphonamide group does not appear to make significant interactions with the protein. Nonetheless the R1 substituent
does affect affinity as its removal (R1 = –H, 7) or replacement with
a methyl (R1 = –CH3, 8) again abrogate activity.
3. Conclusion
A. fumigatus contains five plant-type GH18 chitinases; based on
the structural information for AfChiA1, it is predicted that the acetazolamide binding sites of AfChiA1–5 are identical, suggesting it may
be possible to develop compounds that inhibit all of these enzymes.
We have previously reported various inhibitors of ScCTS1; these
showed different inhibition profiles against AfChiA1; in particular
the binding pocket which accommodated the acetamide group is
much smaller in the case of AfChiA1 compared to ScCTS1. The most
promising inhibitor was acetazolamide. Although acetazolamide
A. W. Schüttelkopf et al. / Bioorg. Med. Chem. 18 (2010) 8334–8340
8337
Figure 2. (A) Structure-based sequence alignment of A. fumigatus ChiA1 and S. cerevisiae CTS1. AfChiA1 secondary structure elements are indicated above the sequence and
labelled. Residue numbers are given for AfChiA1. The ScCTS1 sequence is shaded by sequence similarity between the two enzymes shown (black = identical, grey = chemically
similar residues), while AfChiA1 is shaded by sequence conservation among A. fumigatus ChiA enzymes (purple = 100% identity, then a gradient from blue (mode identical) to
white (less identical)). Residues lining the AfChiA1 active site are highlighted by green filled circles. (B) Acetazolamide (slate) binding to the active site of AfChiA1. The protein
is shown as a grey cartoon with the side chains of active site residues shown as sticks and labelled. Unbiased (i.e., calculated before the addition of the ligand to the model) rAweighted Fo Fc density for acetazolamide contoured at 3.0r is shown in cyan. Possible hydrogen bonds are indicated as black dotted lines, a water participating in indirect
hydrogen bonding between ligand and protein is shown as a red sphere. (C) The active site cavity of AfChiA1 (with bound acetazolamide) coloured by similarity among AfChiA
proteins as described for panel A. Y125, the only non-conserved residue of the acetazolamide-binding site, is labelled.
and various analogues did not show very potent inhibition, they
have relatively low molecular weights. Ligand efficiency is a good
way to characterise how efficiently these core scaffolds bind and
the potential for them to be optimised to low nanomolar compounds.24 Some of the compounds (Table 3) have ligand efficiencies
of better than 0.3 kcal mol1 atom1. Therefore these possess the
potential to be elaborated to compounds with IC50 of <10 nM and
molecular weight of <500, provided good binding interactions are retained. Most of the interactions with the protein are focused around
the amide bond and thiadiazole ring. There is not much scope for
further substitution of the acetyl group as the methyl nearly completely occupies a small pocket. However the sulphonamide does
not appear to make strong interactions and it is possible to replace
this. Therefore optimisation will have to focus on substitution or
replacement of this sulphonamide and enhancement of the interactions of the thiadiazole core with the protein.
4. Experimental
4.1. Expression and purification
AfChiA1 Ser29-Leu335 was expressed and purified as described
previously.21 Briefly, the enzyme was expressed in Pichia pastoris as
a secreted protein. The culture supernatant was subjected to dialysis and concentration, then AfChiA1 was purified using anion exchange chromatography followed by gel filtration. The resulting
pure AfChiA1 protein was used for both kinetic analysis and crystallization trials.
4.2. Crystallisation and structure solution
The protein was concentrated to 36 mg mL1 and crystallized by
hanging drop vapour diffusion as described previously.21 Acetazolamide was incorporated by adding the solid ligand to a crystal-containing drop and incubating for 30 min at room temperature. After
cryoprotection by short immersion in 2.5 M Li2SO4, data were collected at 100 K on beamline ID14-EH3 at the European Synchrotron
Radiation Facility (ESRF, Grenoble, France).
Data were processed and scaled to 2.0 Å using HKL software26
and the structure was solved by molecular replacement with
AMoRe27 using the AfChiA1 apo-structure21 as a search model.
Refinement of the AfChiA1–acetazolamide complex structure proceeded through rounds of minimisation with REFMAC528 and model building with Coot.29 Ligand coordinates and topologies were
generated with PRODRG.30 PyMol31 and ALINE32 were used in the
preparation of Fig. 2.
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Table 3
Activity of compounds investigated
N N
1
R
Compd
1
2
3
4
5
6
7
8
9
10
11
12
13
IC50 (lM)
Hill slope
L.E.
% inhibition at 1 mM
–NHCOCH3
–NHCOCH2CH3
–NHCO(CH2)2CH3
–NHCOCH(CH3)2
–NHCOPh
–NH2
–NHCOCH3
–NHCOCH3
–NHCOCH3
–NHCOCH3
–NHCOCH3
–NHCOCH3
164 ± 75
315 ± 65
>1000
>1000
850 ± 74
>1000
>1000
>1000
730 ± 120
479 ± 210
141 ± 210
243 ± 98
1.1
0.71
0.40
0.34
88
76
33
N/A
51
N/A
25
N/A
64
60
91
76
–NHCOCH3
>1000
N/A
–NHCOCH3
>1000
–NHCOCF3
–NHCOCF3
320 ± 60
>1000
1.2
N/A
–NHCOCF3
>1000
N/A
30
–NHCOCH2CH3
–NH2
–NHCOCH2Cl
–NHCOCH2Cl
–CO(CH2)2CO2H
>1000
>1000
>1000
>1000
>1000
127
410
>1000
N/A
N/A
N/A
N/A
N/A
18
21
N/A
N/A
N/A
R1
R2
–SO2NH2
–SO2NH2
–SO2NH2
–SO2NH2
–SO2NH2
–SO2NH2
–H
–Me
–SH
–Ph
–CF3
–Br
O
14
15
16
R2
S
S
0.23
0.43
0.30
0.44
0.65
22
38
OMe
–Ph
Morpholino
N
17
N/A
0.5
N/A
N/A
N/A
1.1
0.5
1.1
0.7
N
0.26
85
22
Cl
18
19
20
21
22
Allosamidin
8-Chloro-theophylline
Kinetin
–Et
–Me
–CH2CH(CH3)2
–CF3
–CF3
0.12
0.33
L.E. = ligand efficiency in kcal mol1 atom1.24 This was calculated from the equation: DG = RT ln(1/IC50). IC50 standard deviations calculated with 95% confidence limit.
O
H
N
O
S
SO2NH2
HCl (1M), Δ
S
HCl, NH2
N N
SO2NH2
R
Cl
R
N N
96%
TEA / DCM
1
H
N
O
S
SO2NH2
N N
2, R = Et, 13%
3, R = Pr,24%
4, R = i-Pr, 26%
5, R = Ph, 9%
6
O
H2N
S
N N
H
N
Cl
DCM
O
S
H2 N
N N
S
SH
H2SO4
N N
3%
17%
H
N
Ac2O
7
O
S
SH
N N
9
O
S
H2N
N
H
NH2
Cl
rt
23%
H2N
S
N N
19
Scheme 1. Synthesis of acetazolomide analogues.
4.3. AfChiA1 inhibition assays
AfChiA1 activity was assayed in McIlvain’s buffer (pH 5.5).33 The
final reaction mixture consisted of AfChiA1 (10 nM), 0.05 mg/mL
BSA (Thermo) and 4-methylumbelliferyl b-D-N,N0 ,N00 -triacetylchitotrioside (Sigma) (100 lM). Final assay volume was 42 ll in 384-
well black polystyrene plates with a final dimethyl sulphoxide
(DMSO) concentration of 1% in all samples, including controls. Test
and standard compound concentrations ranged from 1000 to
0.15 lM and 10,000 to 1.5 lM, respectively.
Test and standard compounds were placed into columns 1 and
13 of a 384-well polypropylene plate and then serially diluted in
A. W. Schüttelkopf et al. / Bioorg. Med. Chem. 18 (2010) 8334–8340
100% DMSO through half log increments using a JANUS 8-channel
Varispan automated workstation (PerkinElmer). This produced a
compound source plate containing 30 test and 2 standard compounds curves (100 final assay concentration). From this source
plate, 0.42 ll of each compound concentration was then stamped
into replicate black 384-well polystyrene assay plates using a
Hummingbird (Genomic Solutions).
To the assay plates, 20.8 ll AfChiA1 (20 nM) was added to all
wells with the exception the negative controls. The reaction was
initiated by the addition of 20.8 ll of (200 lM) 4-methylumbelliferyl b-D-N,N0 ,N00 -triacetylchitotrioside (stock concentration 200 lM),
both previous additions were executed using a FlexDrop reagent
dispenser (PerkinElmer).
Assay plates were then incubated on a microtitre plate shaker
(Heidolph) at room temperature for 70 min. Fluorescence generated from the release of 4-methylumbelliferone was quantified
using an Envision 2102 Multilabel Reader (PerkinElmer) equipped
with 340 nm excitation (band width 60 nm) and 460 nm emission
(band width 25 nm) filters.
ActivtyBase (Abase) version 5.4 from IDBS was used for the data
processing and analysis. All curve fitting was undertaken using a 4
Parameter Logistic dose–response curve using XLFit 4.2 Model 205.
4.4. Compound Synthesis
4.4.1. Synthesis of 5-amino-2-sulfamoyl-1,3,4-thiadiazole
monohydrochloride (6)
Hydrochloric acid (70 mL, 70.00 mmol, 5.2 equiv) was added to
acetazolamide (2.995 g, 13.34 mmol, 1.0 equiv) and the mixture
stirred for 3 h at reflux. The crude material was purified by column
chromatography (CHCl3/MeOH: 100/0 to 70/30) to yield the product (2.768 g, 96%); mp 179–180 °C; Rf = 0.26 (CHCl3/MeOH: 80/20);
dH (500 MHz, DMSO) 7.91 (2H, br s, NH2-6), 8.09 (3H, s, NH3-7); dC
(125 MHz, DMSO) 157.8 (C-2), 171.6 (C-5); m/z (ES+): 181.1
([M+HCl]+, 100%); HRMS (ES+) 180.9849. ([M+HCl]+ C2H5N4O2S2
requires 180.9848).
4.4.2. Synthesis of 5-propylamido-2-sulfamoyl-1,3,4-thiadiazole
(2)
5-Amino-2-sulfamoyl-1,3,4-thiadiazole
monohydrochloride
(218 mg, 1.01 mmol, 1.0 equiv) was dissolved in DCM (6 mL). Triethylamine (0.30 mL, 2.16 mmol, 2.1 equiv) was added and the
solution stirred for 1.5 h at rt. Then, propionyl chloride (0.20 mL,
2.26 mmol, 2.2 equiv) was slowly added and the mixture left stirring for 1.5 h at rt. Water (1 mL) was added and the precipitate filtered and dried under vacuum. The solid (158 mg) was purified by
column chromatography (CHCl3/MeOH: 100/0 to 78/22) to yield
the product (32 mg, 13%); mp 253–255 °C; Rf = 0.58 (CHCl3/MeOH:
80/20); dH (500 MHz, DMSO) 1.12 (3H, t, H-10, J = 7.5), 2.55 (2H, q,
H-9, J = 7.5), 8.34 (2H, br s, NH2-6), 13.00 (1H, br s, NH-7); dC
(125 MHz, DMSO) 8.8 (C-10), 28.2 (C-9), 161.2 and 164.1 (C-2
and C-5), 173.0 (C-8); m/z (ES+): 237.0 ([M+H]+, 100%), 495.0
([2M+H]+, 71%); HRMS (ES+) 237.0101. ([M+H]+ C5H9N4O3S2 requires 237.0111).
4.4.3. Synthesis of 5-butyramido-2-sulfamoyl-1,3,4-thiadiazole
(3)
5-Amino-2-sulfamoyl-1,3,4-thiadiazole
monohydrochloride
(286 mg, 1.32 mmol, 1.0 equiv) was dissolved in DCM (7 mL). Triethylamine (0.35 mL, 2.51 mmol, 1.9 equiv) was added and the
solution stirred for 1.5 h at rt. Then, butyryl chloride (0.25 mL,
2.36 mmol, 1.8 equiv) was slowly added and the mixture left stirring for 4 h at rt. Water (1 mL) was added and the precipitate filtered and dried under vacuum. The solid (90 mg) was purified by
column chromatography (CHCl3/MeOH: 100/0 to 78/22) to yield
the product (79 mg, 24%); mp 244–246 °C; Rf = 0.54 (CHCl3/MeOH:
8339
80/20); dH (500 MHz, DMSO) 0.91 (3H, t, H-11, J = 7.4), 1.65 (2H,
sext, H-10, J = 7.4), 2.52 (2H, m, H-9), 8.33 (2H, br s, NH2-6),
12.99 (1H, br s, NH-7); dC (125 MHz, DMSO) 13.4 (C-11), 17.9 (C10), 36.7 (C-9), 161.1 and 164.2 (C-2 and C-5), 172.2 (C-8); m/z
(ES+): 251.0 ([M+H]+, 73%); 523.0 ([2M+H]+, 100%); HRMS (ES+)
251.0257. ([M+H]+ C6H11N4O3S2 requires 251.0267).
4.4.4. Synthesis of 5-(2-methyl-propylamido)-2-sulfamoyl1,3,4-thiadiazole (4)
5-Amino-2-sulfamoyl-1,3,4-thiadiazole
monohydrochloride
(274 mg, 1.26 mmol, 1.0 equiv) was dissolved in DCM (7 mL). Triethylamine (0.35 mL, 2.51 mmol, 2.0 equiv) was added and the
solution stirred for 1.5 h at rt. Then, isobutyryl chloride (0.25 mL,
2.34 mmol, 1.9 equiv) was slowly added and the mixture left stirring for 2 h at rt. Water (1 mL) was added and the precipitate filtered and dried under vacuum. The solid was purified by column
chromatography (CHCl3/MeOH: 100/0 to 80/20) to yield the product (90 mg, 26%); mp 254–255 °C; Rf = 0.65 (CHCl3/MeOH: 80/20);
dH (500 MHz, DMSO) 1.16 (6H, d, H-10, J = 6.9), 2.82 (1H, Sept, H-9,
J = 6.8), 8.34 (2H, br s, NH2-6), 13.01 (2H, br s, NH-7); dC (125 MHz,
DMSO) 18.9 (C-10), 33.9 (C-9), 161.3 and 164.3 (C-2 and C-5),
176.1 (C-8); m/z (ES+): 523.0 ([2M+Na]+, 100%), 251.0 ([M+H]+,
33%); HRMS (ES+) 251.0272. ([M+H]+ C6H11N4O3S2 requires
251.0267).
4.4.5. Synthesis of 5-benzylamido-2-sulfamoyl-1,3,4-thiadiazole
(5)
5-Amino-2-sulfamoyl-1,3,4-thiadiazole
monohydrochloride
(315 mg, 1.45 mmol, 1.0 equiv) was dissolved in DCM (7 mL). Triethylamine (0.40 mL, 2.87 mmol, 2.0 equiv) was added and the
solution stirred for 1.5 h at rt. Then, benzoyl chloride (0.30 mL,
2.56 mmol, 1.8 equiv) was slowly added and the mixture left stirring for 2.5 h at rt. Water (1 mL) was added and the precipitate filtered and dried under vacuum. The solid (317 mg) was purified by
column chromatography (CHCl3/MeOH: 100/0 to 78/22) to yield
the product (36 mg, 09%); mp 260–261 °C; Rf = 0.66 (CHCl3/MeOH:
80/20); (found: C, 38.3; H, 3.2; N, 17.9; S, 21.2.
C9H8N4O3S20.7MeOH requires C, 38.0; H, 3.5; N, 18.3; S, 20.9);
dH (500 MHz, DMSO) 7.60 (2H, dt, H-11, J = 7.8, 1.8), 7.71 (1H, tt,
H-12, J = 7.4, 1.2), 8.16 (2H, dd, H-10, J = 8.3, 1.1), 8.38 (2H, br s,
NH2-6), 13.55 (1H, br s, NH-7); dC (125 MHz, DMSO) 128.6 and
128 (C-10 and C-11), 130.9 (C-9), 133.4 (C-12), 162.2 and 164.7
(C-2 and C-5), 165.7 (C-8); m/z (ES+): 285.0 ([M+H]+, 100%), 569.0
([2M+H]+, 20%); HRMS (ES+) 285.0102. ([M+H]+ C9H9N4O3S requires 285.0111).
4.4.6. Synthesis of 5-acetamido-1,3,4-thiadiazole (7)
2-Amino-1,3,4-thiadiazole (161 mg, 1.54 mmol, 1.0 equiv) was
dissolved in DCM (2 mL). Acid chloride (0.15 mL, 2.11 mmol,
1.4 equiv) was slowly added (exothermic), and the mixture stirred
for 5 h at rt. The solution was concentrated after addition of H2O
(1 mL). The crude product was purified by column chromatography
(CHCl3/MeOH: 100/0 to 90/10) to yield the product (38 mg, 17%);
mp 276–277 °C; Rf = 0.83 (CHCl3/MeOH: 80/20); (found: C, 33.9;
H, 3.4; N, 27.6; Cl, 21.8. C4H5N3OS0.03HCl0.18MeOH requires C,
33.5; H, 3.8; N, 28.0; S, 21.4); dH (500 MHz, DMSO) 2.20 (3H, s,
H-8), 9.15 (1H, s, H-2), 12.55 (1H, br s, NH-6); dC (125 MHz, DMSO)
22.4 (C-8), 148.5 (C-5), 158.5 (C-2), 168.6 (C-7); m/z (ES+): 144.0
([M+H]+, 60%), 166.0 ([M+Na]+, 100%); HRMS (ES+) 144.0226.
([M+H]+ C4H6N3OS requires 144.0226).
4.4.7. 5-Acetamido-2-thiol-1,3,4-thiadiazole (9)
5-Amino-1,3,4-thiadiazole-2-thiol
(554 mg,
4.08 mmol,
1.0 equiv), acetic anhydride (1.8 mL, 19.08 mmol, 4.7 equiv) and
concd sulphuric acid (20 mL, 0.37 mmol, 0.09 equiv) were stirred
for 30 min on a steam bath. After cooling, the mixture was
8340
A. W. Schüttelkopf et al. / Bioorg. Med. Chem. 18 (2010) 8334–8340
concentrated under vacuum, and then purified by column chromatography (CHCl3/MeOH: 100/0 to 70/30) to yield the product
(22 mg, 03%); mp 293–295 °C; Rf 0.24 (CHCl3/MeOH: 90/10);
(found: C, 28.1; H, 3.0; N, 22.5; S, 35.1. C4H5N3OS20.3MeOH requires C, 27.9; H, 3.4; N, 22.7; S, 34.7); dY (500 MHz, DMSO) 2.14
(3H, s, H-9), 12.45 (1H, br s, NH-7), 14.06 (1H, br s, SH-6); dC
(125 MHz, DMSO) 22.3 (C-9), 152.2 (C-5), 169;4 (C-8), 183.5 (C2); m/z (ES+): 176.0 ([M+H]+, 100%); HRMS 175.9946. ([M+H]+
C4H6N3OS2 requires 175.9947).
4.4.8. 5-Amino-2-methyl-1,3,4-thiadiazole (19)
Acetyl chloride (0.50 mL, 7.04 mmol, 2.2 equiv) was slowly
added to thiosemicarbazide (291 mg, 3.16 mmol, 1.0 equiv) and
the mixture stirred for 4 h at rt. A solution of NaOH 50% was added
till pH 12–14, and the mixture concentrated under vacuum. The
crude material was purified by column chromatography (CHCl3/
MeOH: 100/0 to 91/09) to yield the product (85 mg, 23%); mp
273–274 °C; Rf = 0.79 (CHCl3/MeOH: 80/20); (found: C, 31.9; H,
4.4; N, 35.4; S, 27.4. C3H5N3S0.05AcOH requires C, 31.5; H, 4.4;
N, 35.6; S, 27.1); dY 500 MHz, DMSO 2.17 (3H, s, H-7), 13.15 (2H,
br s, NH2-6); dC (125 MHz, DMSO) 10.8 (C-7), 148.8 (C-5), 165.8
(C-2); m/z (ES+): 115.8 ([M+H]+, 100%); 253.2 ([2M+Na]+, 100%);
HRMS (ES+) 116.0275. ([M+H]+ C3H6N3S requires 116.0277).
Acknowledgements
This work was supported by a Wellcome Trust Award (Grant
Number 081745). DvA is supported by a Wellcome Trust Senior Research Fellowship. We thank the European Synchrotron Radiation
Facility, Grenoble, for the time at beamline ID14-EH3. The structure has been deposited in the Protein Data Bank (PDB id 2XTK).
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